Project Summary Neisseria gonorrhoeae reproductive tract infections can lead to chronic and severe consequences in women, where diagnosis is frequently delayed by lack of symptoms, contrary to male infections. A once-simple antibiotic treatment gonococcal infection is now compromised by a worldwide increased antibiotic resistance, including the last FDA-approved antibiotic cefixime. The CDC has recently placed N. gonorrhoeae in the urgent threat category of antibiotic resistant microorganisms. There is a strong need for a vaccine against gonorrhea, but this has been delayed by combined lack of successful antigens and animal models of N. gonorrhoeae infections to evaluate protective immunity. Discovery of new bacterial antigens has been enabled by the assumption that pathogens express virulence factors explicitly in vivo. These may have consequently escaped detection in vitro. Based on previous NIH-funded grants (U19 AI084048 and R56 AI107821-01A1), we have examined the global transcriptome of cervico-vaginal lavage and urethral samples from human subjects naturally infected with N. gonorrhoeae attending the National Center for STD Control (NCSTD) in Nanjing, China, by RNA-seq. We reported a differential mRNA expression in these samples as compared to that from each infecting strains grown in vitro, for a large number of genes. We have defined these ?in vivo expressed factors? (IVEFs). Among the IVEFs there are several hypothetical proteins, representing a previously untapped pool of potential new antigens for a N. gonorrhoeae vaccine. These novel IVEFs vaccine candidates have not been examined before because they had not been identified so far. We have used multiple in silico-based predictive criteria to select several hypothetical protein IVEFs to be tested in vitro and in vivo as novel broad vaccine targets. Besides being highly expressed in vivo, the desired features of the selected IVEFs include: broad expression among gonococcal strains; predicted antigenicity; predicted bacterial membrane localization (outer membrane or periplasmic) and surface exposure and lastly, structure features suitable for recombinant production. In addition to predicted antigenicity, some IVEFs share DNA sequence homology to currently explored immunogenic meningococcal vaccine candidates. We will initially focus on 19 IVEFs for recombinant expression and antigenic/immunogenic characterization in mice. Immunogenicity as well as recognition of IVEFs in whole bacteria will be validated using banked hyperimmune sera from subjects naturally exposed to the gonococcus, for which we will use purified IVEFs as well as IVEF over-expression gonococcal mutant strains created in both laboratory strains and clinical isolates from the Nanjing cohort. We will test the ability of mouse immune sera and vaginal lavages as well as banked human sera to induce killing of N. gonorrhoeae in vitro to further establish IVEFs as broad vaccine antigens.